Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5’ long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3’ LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3’LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3’ LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2–12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions.

• page 12, • page 15   Below, please find our answers to all reviewer's comments, presented in the same order as in the report.

Part I -Summary:
This a longitudinal analysis of HIV subtype C LTR diversity and transcriptional activation during acute/early infection in South Africa. The patient population, in vitro methods, and data analysis are well described. Overall, this is a very nice study with interesting findings.

Response:
-We are grateful to the reviewer for their insightful comments on our paper.
Part II -Major Issues: Key Experiments Required for Acceptance:

Response:
-We are grateful to the reviewer for their insightful comments on our paper.

Part III -Minor Issues: Editorial and Data Presentation Modifications:
A few minor revisions would strengthen this manuscript further, including: • Lines 145 and 148 and Table 1: Why is the median square root CD4 T cell count presented rather than the absolute value? Response: An analysis showing the absolute CD4 T cell Count has been included in this article as supporting Table S1 and is referenced on page 8 lines 147 and 151. Furthermore, the following sentence has been added in line 152 -156, "While the median absolute CD4 T cell count was significantly different between the group whose samples were available within a median of 1 day of virus detection and 34 days post infection at the earliest time point (p=0.0240), the median square root CD4 T cell count is reported in this study because a square root transformation has the effect of making the data less skewed and variation more uniform." The title of the supporting table is on page 39 lines 874 -875, " • The diversity inherent in Figure 2 is difficult to see. A phylogram may be better than a circular tree for data visualization / presentation.

Response:
- Figure 2 has been changed to a phylogram to improve data visualization/ presentation. •

Strengths Detailed characterisation of the genetic evolution of the HIV LTR from subtype C strains in the first year after acute infection in 41 patients with a focus on transcriptional activity. Numbers on genetic diversity and evolution are given. A correlation between transcriptional activity and viral loads is demonstrated. Heterogeneous response to LRA activation is shown. No evidence for selection of 4 NF-kappaB sites in the LTR in
South African population in contrast to previous reports from India.

Response:
-We are grateful to the reviewer for their insightful comments on our paper.

Part II -Major Issues: Key Experiments Required for Acceptance:
No additional experiments required.

Response:
-We are grateful to the reviewer for their insightful comments on our paper.

Part III -Minor Issues: Editorial and Data Presentation Modifications:
• Please rephrase line 175-177 on page 6. Not clear what authors want to tell.

Response:
-The sentence on page 10 lines 175 -177 has been paraphrased as follows "The phylogenetic tree shows intra-patient clustering of LTR nucleotide sequences obtained at acute and one-year timepoints and that LTR sequences generated at the acute timepoint, without matching later timepoint samples, were distinct and did not form clusters".

• Line 360: perhaps a number is missing (4 NF-kappa B sites)
Response: -The sentence on page 18 lines 360 -362 has been paraphrased as follows "However, our study did not show that viruses with 4 NF-B binding sites are being preferentially transmitted or have superior transcriptional potential".

Response:
-A manuscript source file is provided and oour manuscript file has been uploaded as a .docx file.
2) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full. At this stage, "All Authors" require contributions. Please ensure that the full contributions of each author are acknowledged in the ""Add/Edit/Remove Authors"" section of our submission form.

Response:
-All copy or trademark symbols have been removed. SuperScript™ has been changed to SuperScript on page 24 line 461, Platinum™ has been changed to Platinum on page 24 line 462, SuperScript™ has been changed to Superscript on page 24 line 470, Platinum™ has been changed to Platinum on page 24 line 470. Figure S3)legend is missing .Please add a full list of legends for your Supporting Information file after the references list.

Response:
-On page 37 lines 874 -889, Figure S3 legend has been added together with the full list (page 37, lines 858 -page 38, line 902) for our Supporting information file.
9) Please make sure that the label in the supplementary legends matches the label in the uploaded Supplementary files (Table S1).

Response:
-The supplementary legends matches the label in the uploaded Supplementary files (Table S1)